The present invention provides, inter alia, antioxidant compositions, including pharmaceutical compositions, of synthetic catalytic cyclic salen-metal antioxidants and reactive oxygen species scavengers for therapy and prophylaxis of disease and prevention of oxyradical-mediated oxidation; methods of using such cyclic salen-metal antioxidants in the prevention and treatment of pathological conditions; methods of using such cyclic salen-metal antioxidants as preservatives and oxyradical quenching agents; methods of using such cyclic salen-metal antioxidants for targeted protection of tissues and/or cell types during cancer chemotherapy; and methods of using such cyclic salen-metal antioxidants to prevent toxicologic damage to individuals exposed to irritating oxidants or other sources of oxidative damage, particularly oxygen-derived oxidative species, such as the superoxide radical and hydrogen peroxide. In addition, the present invention provides compositions and methods that are useful for preventing oxidative damage in human transplant organs and for inhibiting reoxygenration injury following reperfusion of ischemic tissues. In addition, the present invention provides compositions and methods that are useful for chemoprevention of chemical carcinogenesis and alteration of drug metabolism involving epoxide or free oxygen radical intermediates. The present invention also provides novel cyclic salen-metal compounds (CSMCs) having therapeutically useful catalytic properties, and compositions containing such novel compounds.
Molecular oxygen is an essential nutrient for nonfacultative aerobic organisms, including, of course, humans. Oxygen is used in many important ways, namely, as the terminal electronic acceptor in oxidative phosphorylation, in many dioxygenase reactions, including the synthesis of prostaglandins and of vitamin A from carotenoids, in a host of hydroxylase reactions, including the formation and modification of steroid hormones, and in both the activation and the inactivation of xenobiotics, including carcinogens. The extensive P-450 system uses molecular oxygen in a host of important cellular reactions. In a similar vein, nature employs free radicals in a large variety of enzymic reactions.
Excessive concentrations of various forms of reactive oxygen species and of free radicals can have serious adverse effects on living systems, including the peroxidation of membrane lipids, the hydroxylation of nucleic acid bases, and the oxidation of sulthydryl groups and of other sensitive moieties in proteins. If uncontrolled, mutations and/or cellular death result.
Biological antioxidants include well-defined enzymes, such as superoxide dismutase, catalase, selenium glutathione peroxidase, and phospholipid hydroperoxide glutathione peroxidase. Nonenzymatic biological antioxidants include tocopherols and tocotrienols, carotenoids, quinones, bilirubin, ascorbic acid, uric acid, and metal-binding proteins. Various antioxidants, being both lipid and water soluble, are found in all parts of cells and tissues, although each specific antioxidant often shows a characteristic distribution pattern. The so-called ovothiols, which are mercaptohistidine derivatives, also decompose peroxides nonenzymatically.
Free radicals, particularly free radicals derived from molecular oxygen, are believed to play a fundamental role in a wide variety of biological phenomena. In fact, it has been suggested that much of what is considered critical illness may involve oxygen radical (xe2x80x9coxyradicalxe2x80x9d) pathophysiology (Zimmerman, J. J. (1991) Chest 00:1895). Oxyradical injury has been implicated in the pathogenesis of pulmonary oxygen toxicity, adult respiratory distress syndrome (ARDS), bronchopulmonary dysplasia, sepsis syndrome, and a variety of ischemia-reperfusion syndromes, including myocardial infarction, stroke, cardiopulmonary bypass, organ transplantation, necrotizing enterocolitis, acute renal tubular necrosis, and other disease. Oxyradicals can react with proteins, nucleic acids, lipids, and other biological macromolecules producing damage to cells and tissues, particularly in the critically ill patient.
Free radicals are atoms, ions, or molecules that contain an unpaired electron (Pryor, W. A. (1976) Free Radicals in Biol. 1:1). Free radicals are usually unstable and exhibit short half-lives. Elemental oxygen is highly electronegative and readily accepts single electron transfers from cytochromes and other reduced cellular components; a portion of the O2 consumed by cells engaged in aerobic respiration is univalently reduced to superoxide radical (i.e., .O2xe2x88x92) (Cadenas, E. (1989) Ann. Rev. Biochem. 58:79). Sequential univalent reduction of .O2xe2x88x92 produces hydrogen peroxide (i.e., H2O2), a hydroxyl radical (i.e., .OH), and water.
Free radicals can originate from many sources, including aerobic respiration, cytochrome P-450-catalyzed monooxygenation reactions of drugs and xenobiotics (e.g., trichloromethyl radicals, i.e., CCl3., formed from oxidation of carbon tetrachloride), and ionizing radiation. For example, when tissues are exposed to gamma radiation, most of the energy deposited in the cells is absorbed by water and results in scission of the oxygen-hydrogen covalent bonds in water, leaving a single electron on hydrogen and one on oxygen, thereby creating two radicals, i.e., H. and .OH. The hydroxyl radical, i.e., .OH, is the most reactive radical known in chemistry. It reacts with biomolecules, sets off chain reactions and can interact with the purine or pyrimidine bases of nucleic acids. Indeed, radiation-induced carcinogenesis may be initiated by free radical damage (Breimer, L. H. (1988) Brit. J Cancer 57:6). In addition, the xe2x80x9coxidative burstxe2x80x9d of activated neutrophils produces abundant superoxide radical, which is believed to be an essential factor in producing the cytotoxic effect of activated neutrophils. Reperfusion of ischemic tissues also produces large concentrations of oxyradicals, typically superoxide (Gutteridge and Halliwell (1990) Arch. Biochem. Biophys. 283:223). Moreover, superoxide can be produced physiologically by endothelial cells for reaction with nitric oxide, a physiological regulator, forming peroxynitrite, i.e., ONOOxe2x88x92 which may decay and give rise to hydroxyl radical, .OH (Marletta, M. A. (1989) Trends Biochem. Sci. 14:488; Moncada, et al. (1989) Biochem. Pharmacol. 38:1709; Saran, et al. (1990) Free Rad. Res. Commun. 10:221; Beckman, et al. (1990) Proc. Natl. Acad. Sci. (U.S.A.) 87:1620). Additional sources of oxyradicals are xe2x80x9cleakagexe2x80x9d of electrons from disrupted mitochondrial or endoplasmic reticular electron transport chains, prostaglandin synthesis, oxidation of catecholamines, and platelet activation.
Oxygen, though essential for aerobic metabolism, can be converted to poisonous metabolites, such as the superoxide anion and hydrogen peroxide, collectively known as reactive oxygen species (ROS). Increased ROS formation under pathological conditions is believed to cause cellular damage through the action of these highly reactive molecules on proteins, lipids, and DNA. During inflammation, ROS are generated by activated phagocytic leukocytes. As described above; during the neutrophil xe2x80x9crespiratory burst,xe2x80x9d superoxide anion is generated by the membrane-bound NADPH oxidase. ROS are also believed to accumulate when tissues are subjected to ischemia followed by reperfusion.
Many free radical reactions are highly damaging to cellular components, i.e., they crosslink proteins, mutagenize DNA, and peroxidize lipids. Once formed, free radicals can interact to produce other free radicals and non-radical oxidants such as singlet oxygen (1O2) and peroxides. Degradation of some of the products of free radical reactions can also generate potentially damaging chemical species. For example, malondialdehyde is a reaction product of peroxidized lipids that reacts with virtually any amnine-containing molecule. Oxygen free radicals also cause oxidative modification of proteins (Stadtman, E. R. (1992) Science 257:1220).
Aerobic cells generally contain a number of defenses against the deleterious effects of oxyradicals and their reaction products. Superoxide dismutases (SODs) catalyze the reaction:
2.O2xe2x88x92+2H+xe2x86x92O2+H2O2
which removes superoxide and forms hydrogen peroxide. H2O2 is not a radical, but it is toxic to cells and it is removed by the enzymatic activities of catalase and glutathione peroxidase (GSH-Px). Catalase catalyzes the reaction:
2 H2O2xe2x86x922 H2O2+O2
and GSH-Px removes hydrogen peroxide by using it to oxidize reduced glutathione (GSH) into oxidized glutathione (GSSG) according to the following reaction:
2 GSH+H2O2xe2x86x92GSSG+2 H2O2
Other enzymes, such as phospholipid hydroperoxide glutathione peroxidase (PLOOH-GSH-Px), converts reactive phospholipid hydroperoxides, free fatty acid hydroperoxides, and cholesterol hydroperoxides to corresponding harmless fatty acid alcohols. Glutathione S-transferases also participate in detoxifying organic peroxides. In the absence of these enzymes and in presence of transition metals, such as iron or copper, superoxide and hydrogen peroxide can participate in the following reactions which generate the extremely reactive hydroxyl radical, i.e., .OH:
.O2xe2x88x92+Fe3+xe2x86x92O2+Fe2+
H2O2+Fe2+xe2x86x92.OH+OHxe2x88x92Fe3+
In addition to enzymatic detoxification of free radicals and oxidant species, a variety of low molecular weight antioxidants, such as glutathione, ascorb ate, tocopherol, ubiquinone, bilirubin, and uric acid, serve as naturally-occurring physiological antioxidants (Krinsky, N. I. (1992) Proc. Soc. Exp. Biol. Med. 200:248-54). Carotenoids are another class of small molecule antioxidants and have been implicated as protective agents against oxidative stress and chronic diseases. Canfield, et al., (1992) Proc. Soc. Exp. Biol. Med. 200:260, summarize reported relationships between carotenoids and various chronic diseases, including coronary heart disease, cataract, and cancer. Carotenoids dramatically reduce the incidence of certain premalignant conditions, such as leukoplakia, in some patients.
In order to prevent the damaging effects of free radicals and free radical-associated diseases, great efforts have been made to develop new antioxidants that are efficient at removing dangerous oxyradicals, particularly superoxide and hydrogen peroxide, and that are inexpensive to manufacture, stable and possess advantageous pharmacokinetic properties, such as the ability to cross the blood-brain barrier and penetrate tissues. Most recently, Malfroy-Camine, et al. have achieved this goal with their unexpected discovery that members of a class of compounds that were originally described as epoxidation catalysts, the so-called salen-metal complexes, also exhibit potent superoxide dismutase activity and/or catalase activity and, thus, function effectively as catalysts for free radical removal both in vitro and in vivo (see, U.S. Pat. Nos. 5,403,834, 5,834,509, 5,696,109 and 5,827,880, all of which issued to Malfroy-Camine, the teachings of which are incorporated herein by reference). Prior to this discovery, the salen-transition metal complexes had only been described and used as chiral epoxidation catalysts for various synthetic chemistry applications (see, Fu, et al. (1991) J. Org. Chem. 56:6497; Zhang, W. and Jacobsen, E. N. (1991) J. Org. Chem. 56:2296; Jacobsen, et al. (1991) J. Am. Chem. Soc. 113:6703; Zhang et al (1990) J. Am. Chem. Soc. 112:2801; Lee, N. H. and Jacobsen, E. N. (1991) Tetrahedron Lett. 32:6533; Jacobsen, et al. (1991) J. Am. Chem. Soc. 113:7063; Lee, et al. (1991) Tetrahedron Lett. 32:5055).
Malfroy-Camine, et al. have now found that salen-metal complexes are also useful as potent antioxidants for various biological applications, including their use as pharmaceuticals for the prevention and/or treatment of free radical-associated diseases. Pharmaceutical formulations, dietary supplements, improved cell and organ culture media, improved cryopreservation media, topical ointments, and chemoprotective and radioprotective compositions can now be prepared with an effective amount or concentration of at least one antioxidant sal en-metal complex. In addition, Malfroy-Camine, et al. have found that salen-metal complexes can also be used to partially or totally arrest the progression of neurodegenerative diseases. For instance, antioxidant salen-metal complexes can be used for the treatment and prophylaxis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson""s disease, Alzheimer""s disease, etc. Other uses for such salen-metal complexes are disclosed in U.S. Pat. Nos. 5,403,834, 5,834,509, 5,696,109 and 5,827,880.
Although the contributions of Malfroy-Camine, et al. have revolutionized the field of antioxidants that are useful in the prevention and treatment of free radical-associated diseases, it would still be advantageous if salen-metal compounds having increased stability could be developed. The present invention fulfills this and other goals.
It has now been discovered that the stability of salen-metal compounds or, interchangeably, salen-metal complexes can be increased by cyclizing such compounds at the 3,3xe2x80x2-position. As such, in one aspect, the present invention provides cyclic salen-metal compounds having increased stability. In addition, the present invention provides pharmaceutical compositions comprising such antioxidant cyclic salen-metal compounds, therapeutic uses of such antioxidant cyclic salen-metal compounds, and methods and compositions for using such antioxidant cyclic salen-metal compounds in diagnostic, therapeutic and research applications in, for example, human and veterinary medicine.
In one embodiment, the present invention provides cyclic salen-metal compounds having the following general formula: 
In another embodiment, the present invention provides cyclic salen-metal compounds having the following general formula: 
In Formulae I and II, M is a metal, preferably a transition metal, and A is an anion, preferably a halogen or an organic anion (e.g., acetate). Examples of suitable transition metals include, but are not limited to, Mn, Cr, Fe, Zn, Cu, Ni, Co, Ti, V, Ru and Os. Examples of suitable anions include, but are not limited to, PF6, (Aryl)4, BF4, B(Aryl )4, halogen, acetate, acetyl, formyl, formate, triflate, tosylate or, alternatively, the anion can be an oxygen atom typically bound via a double bond to the metal, i.e., M. X1 and X2 are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys. xe2x80x9cY1, Y2, Y3, Y4, Y5 and Y6, in Formulae I and II, are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys. R1, R2, R3 and R4 are independently selected and are functional groups including, but not limited to, hydrogen, halogens, alkyls, substituted alkyls, aryl, substituted aryl, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys; with the proviso that one of R1 or R2 may be covalently linked to one of R3 or R4 forming a cyclic structure. Z, in Formulae I and II, is a bridging group. Q1 and Q2, in Formulae I and II, are independently selected and are functional groups including, but not limited to, hydrogen, halogen, alkyls, substituted alkyls, aryls, substituted aryls, heterocyclics, substituted heterocyclics, heteroaryls, substituted heteroaryls, silyls, aminos, fatty acid esters, alkoxys, aryloxys and acyloxys. The index xe2x80x9cnxe2x80x9d is 0, 1 or 2.
In another aspect, the present invention provides pharmaceutical compositions that have potent antioxidant and/or free radical scavenging properties and function as in vitro and in vivo antioxidants. The pharmaceutical compositions of the present invention comprise an efficacious dosage of at least one species of a cyclic salen-metal complex of Formulae I or II, typically a salen-manganese complex such as a salen-Mn(III) complex. These pharmaceutical compositions possess the activity of dismutating superoxide (i.e., superoxide dismutase activity) and, advantageously, the ability to convert hydrogen peroxide to water and oxygen (i.e., catalase activity). As such, the pharmaceutical compositions of the present invention are effective at reducing pathological damage related to the formation of reactive oxygen species (ROS).
In yet another aspect, the present invention provides methods of using the cyclic salen-metal compounds of the present invention to prevent and/or to treat free radical-associated damage or free radical-associated diseases. More particularly, the present invention provides methods of using cyclic sal en-metal compounds to treat or protect a subject undergoing or expected to undergo: (1) an ischemic episode, such as a myocardial infarction, cerebral ischemic event, transplantation operation, open heart surgery, elective angioplasty, coronary artery bypass surgery, brain surgery, renal infarction, traumatic hemorrhage, tourniquet application; (2) antineoplastic or antihelminthic chemotherapy employing a chemotherapeutic agent that generates free radicals; (3) endotoxic shock or sepsis; (4) exposure to ionizing radiation; (5) exposure to exogenous chemical compounds that are free radicals or produce free radicals; (6) thermal or chemical burns or ulcerations; (7) hyperbaric oxygen; (8) apoptosis of a predetermined cell population (e.g., lymphocyte apoptosis); (9) an inflammatory response; or (10) age-related pathological changes or conditions.
More particularly, the present invention provides methods and compositions for the following: (1) preventing ischemic/reoxygenation injury in a patient; (2) preserving organs for transplant in an anoxic, hypoxic, or hyperoxic state prior to transplant; (3) protecting normal tissues from free radical-induced damage consequent to exposure to ionizing radiation (UV light, gamma radiation, etc.) and/or chemotherapy (e.g., with bleomycin); (4) protecting cells and tissues from free radical-induced injury consequent to exposure to xenobiotic compounds that form free radicals, either directly or as a consequence of monooxygenation through the cytochrome P-450 system; (5) enhancing cryopreservation of cells, tissues, organs, and organisms by increasing the viability of recovered specimens; (6) preventing or treating neurological damage and/or neurodegenerative diseases, and (7) prophylactic administration to prevent, for example, carcino genesis, cellular senescence, cataract formation, formation of malondialdehyde adducts, HIV pathology and macromolecular crosslinking, such as collagen crosslinking.
Other features, objects and advantages of the invention and its preferred embodiments will become apparent from the detailed description which follows.